1. Field of the Invention
This invention is in the field of solid-phase peptide synthesis. More specifically, this invention relates to a novel computer-driven amino acid indexer for rapidly synthesizing peptide samples on a solid support with very high amino acid sequence accuracy.
2. Description of the Related Art
Solid-phase methods of peptide synthesis have been developed in which proteins of defined amino acid sequence are prepared by the step-wise addition of amino acids in the defined order to a growing peptide chain on a solvent-resistant matrix. De novo design and synthesis of peptides has become an indispensable technique in scientific research for a variety of important reasons.
The design and synthesis of novel and naturally occurring peptides makes it possible to study protein topologies and conformations as well as the kinetics of protein folding. Additionally, many biologically active substances, such as hormones and signal molecules, are peptides. Further peptide synthesis is useful in identifying the receptor sites on cell surfaces where hormones and signal molecules are active. There is keen interest in this field because synthetic peptides may be useful as pharmaceutical agents with a variety of applications, such as vaccines effective against bacterial and viral infections. And synthetic peptides may also be used to identify specific antibody binding sites, known as epitopes, on antigens, disease-inducing foreign organisms or viruses. As a corollary to this last field of endeavor, it is also possible to use synthetic peptides as antigens or as fragments which are effective epitope mimics to stimulate the production of particular antibodies by the host organism (Scientific American Feb. 1983:48-56; Nature 306:9 [1983]).
The overall immune systems of higher animals comprise very complex interactions of non-specific defensive scavengers (phagocytes), specific defenders (i.e., antibodies), mediators, and modulators. The capability of higher animals to fight a given disease is greatly dependent on the ability of the host's antibodies to recognize antigens and bind them tightly and specifically. This binding activity precipitates a sequence of events that leads to the neutralization and elimination of the organism or virus responsible for the disease. Diseases which are subject to surveillance by the host immune system in this general manner include, by way of example, influenza, tetanus, polio, smallpox, and many cancers. Vaccines and therapeutic compounds are being sought for some cancers, acquired immunodeficiency syndrome (AIDS), hepatitis B, herpes and other diseases. The effects of certain chemical warfare agents are also being studied to determine whether such agents are subject to resistance by antibodies according to the general scheme described herein.
A particularly challenging endeavor in the field of immunology is to characterize mechanism and binding specificity of a host's immune response to a particular peptidic antigen. Each antigen binding site, or epitope, is specific to a particular antibody. Solid-phase peptide synthesis techniques are especially useful in this endeavor, in that a synthetic peptide having the same amino acid sequence as an epitope, or one so similar that it effectively mimics the epitope, will bind an antibody in the same tight and specific manner as will a natural antigen possessing the epitope. Peptide fragments which copy of mimic an epitope can trigger the same immune response as a peptidic antigen containing the epitopic center as well as the genetic material required to replicate itself. Thus, immunization by using peptide fragments substantially reduces the risk of infection or deleterious side effects. By making up sample peptides and mapping their antibody binding characteristics through analytical techniques such as enzyme-linked immunosorbent assay (ELISA) or radioimmuno assay (RIA), it is possible to characterize the antibody reactivity of a large number of peptides and identify those which are active as epitopic centers.
The antibody reactivity mapping technique may be used in conjunction with a technique recently introduced by Hendrik Mario Geysen for synthesizing and testing large numbers of peptides (U.S. Pat. No. 4,833,092, May 23, 1989, Method for Determining Mimotopes, and U.S. Pat. No. 4,708,871, Nov. 24, 1987, Antigenically Active Amino Acid Sequences). In this technique, peptides are synthesized incrementally backwards according to processes known in the art of peptide synthesis chemistry. An activated derivative of the correct individual amino acid is pipetted onto each growing peptide chain, each peptide being synthesized separately on a plastic pin. An 8.times.12 or similar matrix of pins may be used in the syntheses. After hundreds of peptides are individually prepared, each on its own respective pin, the pins are assayed by ELISA for antibody reactivity. The peptides made according to this method are typically eight amino acid residues in length and have an overlapping pattern along the entire protein sequence. (The testing of an antigen containing 100 residues in a defined sequence according to the overlapping pattern would require synthesizing eight-residue test samples, beginning with one corresponding to residues 1-8 of the antigen, the next corresponding to residues 2-9 of the antigen, then 3-10, through the last peptide which would correspond to residues 93-100 of the antigen).
Since the pioneering work of R. Bruce Merrifield in developing a solid-phase peptide synthetic method using solvent-resistant polystyrene beads, Science 232:341-347 [1986], techniques of peptide synthesis have remained essentially labor-intensive and time-consuming. This means that skilled technicians must spend long periods of time in performing mechanical tasks, such as pipetting amino acid derivatives into a matrix of solvent-resistant wells for incremental growth of peptide chains in desired sequences. Matching the properly ordered amino acid with the proper well in the matrix has remained essentially a manual task. With current techniques, even an experienced technician can suffer from mental fatigue and confusion during synthesis of multiple peptide samples, one hundred to one thousand, for example, while trying to keep the different wells identified and the prescribed amino acid sequences straight. Notwithstanding the experience and skill required of the technicians so employed and the time that such mechanistic processes inherently require, error rates have typically exceeded the rates that are normally considered acceptable in a biochemistry laboratory. It is apparent that committing trained and skilled technicians to laborious and tedious occupations is inefficient and undesirable.
Given the keen interest in peptide synthesis and analysis shown by government, industry, and academic institutions, it is highly apparent that simplified means for peptide synthesis which overcome the technical problems associated with current techniques are needed. Simplification can reduce technical errors in the sequencing of amino acids during synthesis, save time, help to reduce human error evident in tasks such as the set up of synthetic apparatus, and free technical personnel for other tasks. In this invention, an amino acid indexer for peptide synthesis is provided which simplifies the various technical difficulties associated with peptide synthesis and renders the process more efficient by saving time, reducing errors, and lowering the level of skill absolutely required to perform the mechanical functions of peptide synthesis.